Inhibition of Resistant Variants of HIV Protease The AIDS epidemic encompasses more than 30 million people infected with HIV. Patient outcomes were improved dramatically by highly active antiretroviral therapy including inhibitors of the vira protease. No cure exists, however, and the long term effectiveness of current AIDS therapy is confronted by the challenge of rapid evolution of drug- resistant HIV. Hence, there is urgent need for new therapies to overcome the problem of drug-resistance. The potent drug darunavir targets resistant protease variants, and our crystal structures defined how darunavir binds HIV protease. Despite darunavir's high potency, resistance has arisen in the clinic to all current drugs. The appearance of new resistance mutations, diverse resistance mechanisms and adverse side effects necessitate the development of novel inhibitors to expand the repertoire and potency of antiviral agents for resistant HIV. Our studies of structures and activities of protease mutants have identified distinct molecular mechanisms for resistance including mutations that: 1) decrease protease interactions with inhibitors; 2) decrease the enzyme stability; or 3) increase the flap mobility. These insights and the strategy of incorporating more interactions with the protease backbone have led to a series of novel antiviral inhibitors with excellent potency for resistant HIV. During this project period we have identified a unique resistance mechanism due to mutation L76V and discovered extremely resistant protease variants that evade inhibition at the autoprocessing stage, unlike the wild type protease precursor. Our X-ray structures have guided the design of novel inhibitors with 10-fold greater antiviral potency than darunavir for resistant viral strains, as well as inhibitors 10-fold more effective than darunavir against highly resistant proteases. Our proposed studies will focus on discovery of the unique molecular mechanisms for high level resistance to protease inhibitors and the application of these insights to design the next generation of antiviral inhibitors. These multidisciplinary studies leverage the expertise, unique resources and novel approaches developed in the PIs groups together with an established set of collaborators to integrate computational, X-ray crystallographic, biochemical and biophysical techniques with inhibitor design, chemical synthesis, and virology studies. The expected outcomes will be 1) accurate predictions for resistance, 2) discovery of novel and conserved molecular mechanisms for resistance, and 3) new antiviral inhibitors for resistant HIV infections.